Title (Aim):
To separate and identify the components of a mixture of amino acids using paper chromatography.
Theory:
Chromatography is a powerful analytical technique used to separate the components of a mixture based on their differential affinities towards a stationary phase and a mobile phase. In paper chromatography, the stationary phase is a sheet of cellulose-based filter paper, and the mobile phase is a suitable solvent or solvent mixture. When a mixture is spotted onto the paper and developed in the solvent, its components travel at different rates depending on their solubility and interaction with the stationary phase. This technique is especially useful in the separation of biomolecules such as amino acids, sugars, and plant pigments.
Principle:
Paper chromatography works on the principle of partition chromatography, where the components of the mixture partition between two phases: the stationary phase (water molecules bound to cellulose in the paper) and the mobile phase (organic solvent). Components with higher affinity for the mobile phase travel faster and farther on the paper, whereas those with higher affinity for the stationary phase travel slower. The separated components can be visualized using specific reagents such as ninhydrin for amino acids. The Rf value (Retention factor) of each component can be calculated to help in its identification.
Requirements:
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Chromatography paper (Whatman No. 1 filter paper)
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Amino acid mixture (e.g., glycine, alanine, serine)
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Individual amino acid standards (optional)
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Capillary tubes or micropipette
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Chromatography jar or glass beaker with lid
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Solvent system (e.g., n-butanol:acetic acid:water in 4:1:5 ratio)
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Pencil and ruler
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Ninhydrin solution (0.1% in acetone or ethanol)
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Spray bottle or cotton swab
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Hot air oven
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Forceps and gloves
Procedure:
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Cut a strip of chromatography paper approximately 20 cm long and 5 cm wide.
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Using a pencil (not pen), draw a horizontal line 2 cm from the bottom edge. This is the baseline.
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Mark 3-4 points equally spaced along the baseline to spot the samples (e.g., sample mixture and standard amino acids).
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Using a capillary tube or micropipette, apply small spots of the amino acid samples on the marked positions. Allow each spot to dry before reapplying to concentrate the sample.
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Prepare the chromatography jar by adding about 1 cm depth of the solvent system and cover the jar to allow solvent vapor equilibration.
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Once the sample spots are dry, suspend the chromatography paper vertically in the jar so that the bottom edge is immersed in the solvent but the spots remain above the solvent level.
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Close the jar and allow the solvent to rise up the paper undisturbed for about 45–60 minutes or until it is about 2–3 cm from the top.
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Remove the paper carefully, mark the solvent front with a pencil immediately, and allow the paper to dry.
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Spray the paper evenly with ninhydrin solution in a fume hood or use a cotton swab to apply it over the chromatogram.
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Dry the paper using a hot air oven (or hair dryer) at 60–70°C for 5–10 minutes to visualize the amino acid spots, which appear as purple or blue-violet stains.
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Measure the distance traveled by each spot and the solvent front from the baseline using a ruler.
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Calculate the Rf values using the formula:
Observation:
| Spot No. | Sample Name | Distance travelled by spot (cm) | Distance travelled by solvent front (cm) | Rf value |
|---|---|---|---|---|
| 1 | Amino acid A | X₁ | Y | X₁/Y |
| 2 | Amino acid B | X₂ | Y | X₂/Y |
| 3 | Amino acid mixture | X₃, X₄, ... | Y | X₃/Y, X₄/Y |
Result:
The paper chromatogram showed distinct spots for different amino acids present in the mixture. The calculated Rf values matched with those of standard amino acids, confirming their identity.
Conclusion:
Paper chromatography successfully separated and identified the amino acids in the mixture based on their differential solubilities and interactions with the mobile and stationary phases.
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